Different mechanisms of acquired resistance to fluorinated pyrimidines in human colorectal cancer cells

5-Fluorouracil (5-FU), 5-fluoro-2'-deoxyuridine (FdUrd) and 5-trifluorothym idine (F(3)dThd) are antimetabolites which are metabolized to their corresp onding active forms which inhibit DNA synthesis via inhibition of thymidyla te synthase (TS). To investigate ways of overcoming 5-FU-resistance, we est ablished acquired-resistant colorectal cancer cell lines against these thre e drugs by continuous and stepwise escalation of drugs, and analyzed the cy totoxicity and the mechanism of resistance to the drugs. When cells were in cubated with the 3 drugs for 72 h, the resistance ratio to parental DLD-1 h uman colorectal tumor cells was 65.2 for DLD-1/5-FU, 9.7 for DLD-1/FdUrd an d 448.6 for DLD-1/ F(3)dThd cells. DLD-1/5-FU cells did not show any cross- resistance against FUrd and F(3)dThd. However, DLD-1/FdUrd cells showed 3- and 9-fold increased resistance to 5-FU and F(3)dThd, respectively, and DLD -1/F(3)dThd cells also showed about 90-fold resistance to FdUrd. Analysis o f enzyme activities and gene expression associated with pyrimidine metaboli sm indicated that a significant decrease in orotate phosphoribosyltransfera se activity in DLD-1/5-FU cells, a 7-fold increase of TS mRNA in DLD-1/FdUr d cells, and a 37-fold decrease in thymidine kinase activity of DLD-I/ F(3) dThd cells were the major mechanisms of drug resistance. These findings wer e closely associated with the cytotoxicity of 5-FU, FdUrd and F(3)dThd agai nst the established 5-FU-, FdUrd- or F(3)dThd-resistant cells. When DLD-1/F dUrd cells expressing increased TS mRNA were treated with FdUrd and F(3)dTh d for only 4 h, the resistance ratios of DLD-1/FdUrd cells to parental DLD- 1 cells were markedly different for FdUld and F(3)dThd, suggesting that the cytotoxicity with short-time exposure to F(3)dThd is due to a mechanism ot her than TS inhibition, although the cytotoxicity of F(3)dThd in the short- time is low compared to that of long-time exposure. In conclusion, F(3)dThd , an antimetabolite that inhibits TS activity, may be effective against 5-F U and/or FdUrd-resistance in colorectal cancer cells caused by amplificatio n of TS and/or deletion of orotate phosphoribosyltransferase.